The accurate labeling of meat products is mandated and monitored by the United States Department of Agriculture (USDA) as well as by state and local governments. Mixing undeclared species in meat products is illegal under Food Labeling Regulations. Apart from regulatory reasons, the prevention of adulteration of meat products with less desirable meat species is important to the consumers for economic, health, food safety and religious reasons.
Several methods have been developed to identify meat species including electrophoresis, chromatography, DNA hybridization, and immunoassays. Immunological techniques, including agar-gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) are most commonly applied for meat species identification.
There are several disadvantages to the official method AGID. Concentrated antiserum preparations are required to obtain visible precipitin lines in AGID. Obtaining the antiserum is expensive in large-scale testing. Furthermore, the sensitivity of AGID is variable. Usually ten percent or more contamination must be present to detect adulteration with this method. Lastly, AGID cannot be used for species identification in cooked meat because of the shortage of commercial antiserum specific to cooked meats.
The ELISA method has emerged as a sensitive, rapid, and specific method for meat speciation. This method can detect levels as low as one percent or less of meat contamination. Furthermore, it requires only simple sample preparation and no expensive equipment or highly skilled operator. In addition, the ELISA technique can be used for both qualitative and quantitative analysis of meat proteins.
Specific antibodies, either polyclonal antibodies or monoclonal antibodies (MAbs), are required as capture reagents in ELISA. Most of the ELISA methods currently employed for meat species identification use polyclonal antibodies as a capture reagent. However, polyclonal antibodies have disadvantages such as limited production, variable affinity and a requirement for further purification to eliminate cross-reaction.
Using MAb based ELISA can provide better data quality and eventually reduce the cost of assays. To date, the monoclonal antibodies which are utilized in ELISA based assays for meat identification have been raised against native proteins. The antibodies are useful for the detection of meat in raw meat mixtures.
A few disclosures of the production of polyclonal antibodies for the qualitative detection of meat species in cooked meat samples has been reported. These polyclonal antibodies have been raised against native heat-resistant immunizing antigens.
Wide spread meat species adulteration has been found particularly among heat-processed meat products and retail meat markets. Hsieh et al. (1995) Jrnl. of Food Quality 19:1-13, indicated that the violation rate in cooked products was higher than raw meats (22.9% versus 15.9%) in the domestic market. This adulteration can have serious health consequences. Poultry carries pathogens that cause food-borne diseases. Cooking poultry requires a minimum internal temperature of 71.1° C. (160° F.) to kill salmonella. Beef requires a minimum internal temperature of 68.3° C. (155° F.) to kill E. Coli H7:0157. If a meat product like beef is contaminated with poultry meat, it might impose a potential health hazard due to the inadequate heat-processing even at a very low level of contamination.
Additionally, regulatory controls to prevent the spread of BSE have prohibited the use of certain animal proteins in feed in several countries, requiring accurate analytical methods for detecting prohibited material in feed stuffs to ensure compliance with the new regulations. Microscopic analysis is commonly used to identify constituents of animal origin, e.g. blood clots, bone fragments, feather fragments, hair or muscle fiber, in feed stuffs. Hahn (1999) Naturwissenschaften 86:62-70. The method is simple and inexpensive, although it requires a certain degree of expertise to distinguish bone fragments of mammals from those of poultry and fish by observing the shape of lacunae. Methods based on PCR amplification of mitochondrial DNA have recently been developed, but generally are not able to distinguish between different tissues of the same species. Immunological methods have played a central role in the species identification of raw and heat processed meats. Hsieh et al., “Monoclonal antibodies against heat-treated muscle proteins for the species identification and endpoint temperature determination of cooked meats” Quality attributes of muscle foods, 1999, pp. 287-307, Plenum Publishing Co., New York, N.Y. The official enzyme immunoassay method for analyzing mammalian proteins of rendered animal material in the UK utilizes antisera raised against heat-stable proteins. Ansfield (1994) Food & Agricultural Immunology, 6:419-433. However, it detects most mammalian proteins without discrimination between prohibited and allowed proteins and requires the complement of microscopic analysis to confirm the presence of mammalian MBM (meat and bone meal).
Thus, development of a suitable screening method to detect undeclared exogenous meat is important to comply with the food labeling regulation and animal feed regulation. There is, therefore, needed a method for the determination of species identification in cooked meats, as well as in animal feed.
In our experience, crude thermostable immunogens could theoretically be used in the production of species-specific MAbs, but were actually ineffective because the chance of success in selecting a specific clone by screening with crude antigen is remote. The use of a defined antigen with known antigenic determinants, a so-called species marker, would substantially increase the chance of eliciting specific antibodies. Thus, besides thermostability, the selected species marker needs to possess a unique antigenic region for a given species that is not present in the counterpart molecules of other species.